The present invention relates to rhesus monkey (Macaca mulatta) DNA molecules encoding the melanocortin-4 receptor protein belonging to the rhodopsin sub-family of G-protein coupled receptors, recombinant vectors comprising DNA molecules encoding rhesus MC-4R, recombinant host cells which contain a recombinant vector encoding rhesus MC-4R, the rhesus MC-4R protein encoded by the DNA molecule, and methods of identifying selective agonists and antagonists of rhesus MC-4R.
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCRs). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as xcex1-, xcex2, or xcex3-melanocyte stimulating hormones (xcex1-, xcex2-, xcex3-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
U.S. Pat. No. 5,532,347 (issued Jul. 2, 1996) to Cone and Mountjoy discloses human and mouse DNA molecules which encode MC-1R (also known in the art as xcex1-MSH-R). The expressed human protein contains 317 amino acids.
U.S. Pat. No. 5,280,112 (issued Jan. 18, 1994) and U.S. Pat. No. 5,554,729 (issued Sep. 10, 1996), both to Cone and Mountjoy, disclose human and mouse DNA molecules which encode MC-2R (also known in the art as ACTH-R). The human MC-2R protein contains 297 amino acids.
Mountjoy, et al. (1992, Science 257: 1248-1251) describe DNA molecules and the concomitant protein for human MC-1R and human MC-2R.
Chhajlani, et al. (1992, FEBS Letters 309: 417-420) also disclose a human DNA molecule comprising an open reading frame which encodes human MC1-R.
Roselli-Rehfuss, et al, (1993, Proc. Natl. Acad. Sci 90: 8856-8860) disclose a cDNA clone encoding rat MC-3R cDNA.
U.S. Pat. No. 5,622,860 (issued Apr. 22, 1997) and U.S. Pat. No. 5,703,220 (issued Dec. 30, 1997) to Yamada and Gantz, disclose DNA molecules which encode human MC-3R and human MC-4R, respectively (see also Gantz, et al., 1993, J. Biol. Chem. 268(11): 8246-8250).
A DNA molecule encoding human MC-5R was also disclosed by Mountjoy, et al. (1994, Mol. Endocrin. 8: 1298-1308).
Chhajlani, et al. (1993, Biochem. Biophys. Res. Comm., 195(2): 866-873) disclose a DNA molecule which the authors state encodes MC-5R. This clone was initially designated MC2.
Fathi, et al. (1995, Neurochemical Research 20(1):107-113) also disclose a DNA molecule thought to encode human MC-5R. There are several sequence discrepancies when compared to the DNA molecule disclosed by Chhajlani, et al., id.
Griffon, et al. (1994, Biochem. Biophys. Res. Comm., 200(2): 1007-1014) disclose DNA clones from human and rat which encode MC-5R. The human DNA sequence agrees with the human DNA sequence disclosed in Fathi et al. id.
Gantz, et al. (1994, Biochem. Biophys. Res. Comm., 200(3): 11214-11220; see also U.S. Pat. No. 5,710,265, issued Jan. 20, 1998 to Yamada and Gantz) and Labbe, et al. (1994, Biochemistry 33: 4543-4549) disclose DNA clones from mouse which encode MC-5R.
Barren, et al. (1994, J. Mol. Endocrin. 12: 203-213) disclose DNA clones from sheep which encode MC-5R.
In rodents, MC-4R has been implicated as a key regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997, Nature 385: 165-168) and with a MC-4R knock-out mouse (Huszar et al., 1997, Cell 88: 131-141).
Compounds that bind to such receptors were previously identified by binding to human and/or rodent receptors and evaluated for their efficacy in rodents. However, the neuroendocrine process can differ between rodents and man. It is also expected that some compounds exhibit different binding affinities for different species homologues of the same receptor (Fong et al., 1992, J.Biol. Chem. 267:25666-25671; Hartig et al., 1992, TIPS 13:152-159).
Before compounds can be selected as a drug candidate it is first evaluated for a physiological effect in rodents and then in the rhesus primate. It is often that one compound may be effective in one animal species but not in another. Previously, it has been impossible to determine if the failure was due to an altered melanocortin pathway in different species, or due to a compounds having a lower affinity for one particular species. Past protocols required the use of a rhesus brain membrane to determine the in vitro biochemical activity of compounds, if such protocol could be successfully employed.
It is desirable to correlate in vivo data with in vitro biochemical activity of compounds.
It is also desirable to first select compounds that are active for the rhesus receptor in vitro.
It is also desirable to identify compounds which can determine the relevance of receptor targets in rhesus and allow selection of novel drugs to treat obesity.
It is further desirable to discover new drugs which effect pathophysiological processes by modulating the effects in rhesus to identify melanocortin active process in primates, followed by human clinical trials.
The present invention addresses and meets these needs by disclosing an isolated nucleic acid fragment which expresses a form of rhesus MC-4R, recombinant vectors which house this nucleic acid fragment, recombinant host cells which expresses rhesus MC-4R and/or a biologically active equivalent, and pharmacological properties of this rhesus MC-4R protein.
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel rhesus monkey (Macaca mulatta) melanocortin-4 receptor (rhMC-4R). The nucleic acid molecules of the present invention are substantially free from other nucleic acids.
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes MRNA which expresses a novel rhesus MC-4R, this DNA molecule comprising the nucleotide sequence disclosed herein as SEQ ID NO:1.
The present invention also relates to biologically active fragments or mutants of SEQ ID NO:1 which encodes mRNA expressing a novel rhesus MC-4R. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of a wild-type MC-4R protein, including but not limited to the rhesus MC-4R receptor protein as set forth in SEQ ID NO:2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-4R function.
A preferred aspect of this portion of the present invention is disclosed in FIG. 1, a rhesus cDNA molecule encoding a novel MC-4R (SEQ ID NO:1).
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to subcellular membrane fractions of the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) which contain the proteins encoded by the nucleic acids of the present invention. These subcellular membrane fractions will comprise either wild-type or mutant forms of rhesus melanocortin-4 receptor proteins at levels substantially above endogenous levels and hence will be useful in various assays to select modulators of rhesus melanocortin-4 receptor protein as described throughout this specification.
The present invention also relates to a substantially purified form of the rhesus melanocortin-4 receptor protein, which comprises the amino acid sequence disclosed in FIG. 2 and set forth as SEQ ID NO:2. A preferred aspect of the present invention is disclosed in FIG. 2 and is set forth as SEQ ID NO:2, the amino acid sequence of the novel rhesus melanocortin-4 receptor protein.
The present invention also relates to biologically active fragments and/or mutants of the rhesus melanocortin-4 receptor protein comprising the amino acid sequence set forth as SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-4R function.
The present invention also relates to assays to screen or select for various modulators of MC-4R activity, methods of expressing the MC-4R protein and biological equivalents disclosed herein, recombinant host cells which comprise DNA constructs which express these receptor proteins, and compounds identified through these assays which act as agonists or antagonists of MC-4R activity.
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type vertebrate MC-4R activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase (GST)-MC-4R fusion constructs which include, but are not limited to, either the intracellular domain of rhesus MC-4R as an in-frame fusion at the carboxy terminus of the GST gene, or the extracellular and transmembrane ligand binding domain of MC-4R fused to the amino terminus of GST, or the extracellular and transmembrane domain of MC-4R fused to an immunoglobulin gene by methods known to one of ordinary skill in the art. Soluble recombinant GST-MC-4R fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen).
Therefore, the present invention relates to methods of expressing the rhesus MC-4R protein and biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these receptor proteins, and compounds identified through these assays which act as agonists or antagonists of MC-4R activity.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the rhesus form of MC-4R, or a biologically active fragment thereof.
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of rhesus MC-4R, or rhesus fragments MC-4R fragments, mutants or derivatives of SEQ ID NO:2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode MRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for vertebrate MC-4R function.
It is a further object of the present invention to provide the rhesus MC-4R proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding rhesus MC-4R or a biological equivalent thereof.
It is an object of the present invention to provide a substantially purified form of the rhesus MC-4R protein, as set forth in SEQ ID NO:2.
It is an object of the present invention to provide for biologically active fragments and/or mutants of the rhesus MC-4R protein, such as set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is also an object of the present invention to provide for MC-4R-based assays to select for modulators of this receptor protein. These assays are preferably cell based assays whereby a DNA molecule encoding MC-4R is transfected or transformed into a host cell, this recombinant host cell is allowed to grow for a time sufficient to express MC-4R prior to use in various assays described herein.
It is a further object to provide for membrane preparations from host cells transfected or transformed with a DNA molecule encoding MC-4R for use in assays to select for modulators of MC-4R activity.
It is also an object of the present invention to provide for MC-4R-based in-frame fusion constructions, methods of expressing these fusion constructs, biological equivalents disclosed herein, related assays, recombinant cells expressing these constructs, and agonistic and/or antagonistic compounds identified through the use of the nucleic acid encoding vertebrate MC-4R protein as well as the expressed protein.
As used herein, xe2x80x9crhxe2x80x9d or refers toxe2x80x94rhesusxe2x80x94.
As used herein, xe2x80x9cMC-4Rxe2x80x9d refers toxe2x80x94melanocortin 4 receptorxe2x80x94.
As used herein, xe2x80x9cGPCRxe2x80x9d refers toxe2x80x94G-protein coupled receptorxe2x80x94.
Whenever used herein, the term xe2x80x9cmammalian hostxe2x80x9d will refer to any mammal, including a human being.